In many presently used bioassays, a molecule, such as DNA or a protein, is immobilized onto a substrate. For example, Western blots immobilize a protein onto a substrate, such as PVDF or nitrocellulose membrane, which is then probed with a ligand to the protein, such as an antibody, to determine whether the protein is present in a sample. Furthermore, Southern and Northern blots utilize a similar process, wherein DNA and RNA respectively are immobilized onto a nitrocellulose substrate, and probed with nucleic acid molecules having a complementary sequence to the desired DNA or RNA. In yet another example, an enzyme-linked immunosorbent assay (ELISA) involves immobilizing an antibody onto a substrate, and then exposing the immobilized antibody to a sample which is suspected of containing the antigen. If the antigen is present, it will bind to the immobilized antibody, and that binding can subsequently be detected. The methods described above employ primarily hydrophobic interactions between the molecule and substrate to immobilize the molecule. Hence, successful use of these techniques is dependent upon the chemical properties of the substrate.
Although these methods are widely used and accepted, they contain inherent limitations which can be detrimental to the productivity of researchers. For example, immobilizing a molecule onto a substrate is a very time consuming process. Initially, the molecules must electrophoresed using SDS-PAGE or agarose gel electrophoresis, which involves pouring the gel (and in the case of SDS-PAGE, exposing the researcher to toxic bis-acrylamide), installing the gel into a cassette, preparing numerous buffers, preparing the samples for electrophoresis, loading the samples onto the gel, and then electrophoresing the samples.
Immediately after the sample has been electrophoresed and molecules of the sample separated by size, the molecules must be transferred to a substrate, and immobilized onto the substrate. This immobilization involves the preparation of numerous buffers, careful handling of the substrate and gel, and carefully contacting the substrate to the gel in the presence of a buffer to avoid the entrapment of air bubbles between the gel and the substrate. A force, such as an electric current is then used to transfer molecules from the gel to the substrate.
More recently, efforts have been made to produce chips upon which molecules such as proteins or DNA are immobilized in predetermined arrays. Patterning such molecules on semiconductor substrates, coupled with specific recognition, are essential for the realization of bio-molecular networks. Furthermore, addressable arrays of DNA [Fodor, S. P. A., Read, J. L., Pirrung, M. C., Stryer, L., Lu, A. T., and Solas, D. Science 251:767 (1991); and Southern, E. M. Trends in Genetics 12:110 (1996) both of which are hereby incorporated by reference in their entireties] or proteins immobilized on a substrate can be used to provide tools for information retrieval, hybridization of DNA and binding affinity for molecules such as proteins, antibodies, lipids or carbohydrates in quick and reliable manner.
One method of producing such chips borrows substantially from photolithographic microfabrication techniques developed and optimized by the computer microprocessor industry, which permit the economic production of large batches of chips using photographic templates. More specifically, current lithographic approaches for immobilizing molecules, particularly biomolecules onto a substrate, use chemical methods to specifically treat substrates with photoresist and then using photomasking, a light beam or an electron beam to define the pattern of immobilization of the molecule on the substrate. Examples of the use of this technique include microlithography on self-assembled monolayers and lipids, microcontact printing, microfluid networks and light directed combinatorial synthesis [Prime, K. L., and Whitesides, G. M. Science 252:1164 (1991); Dulcey, C. S. et al., Science 252:551 (1991); Berggren, K. K., et al., Science 269:1255 (1995); Jackman, R. J.,et al., Science 269:664 (1995); Healey, B. G., et al., Science 269:1078 (1995); Delamarche, E., et al., Science 276:779 (1997); Groves, J. T., et al., Science 275:651 (1997); and Burke, D. T., et al., Genome Research 7:189 (1997), all of which are hereby incorporated by reference in their entireties].
However, all of the above mentioned approaches contain inherent limitations in that they all depend either on photolithographic techniques or substrate chemistry. For example, a proper control of the substrates enables the sequential synthesis of oligonucleotides [Fodor, 1991].
Hence, what is needed is an apparatus for immobilizing molecules, such as biomolecules, onto a substrate which permits patterning of the immobilization of molecules in adressable arrays, so that the substrates produced with the apparatus have ready applications in the production of bio-molecular networks, and to provide tools for information retrieval, hybridization of DNA and binding affinity of ligands for molecules, such as proteins, DNA, RNA, lipids, or carbohydrates immobilized on the substrate.
What is also needed is a method of immobilizing a molecule onto a substrate reliably and economically, and is not dependent upon the chemistry of the substrate.
The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.